The long-term objective of this research program is to develop novel types of phosphoramidate antitumor agents directed against specific cellular targets. Chemical mechanistic information discovered in earlier years of this project will be utilized to guide the design, synthesis, and evaluation of these new mechanism-based phosphoramidates. Specific aims include: 1) Design and synthesis of phosphoramidate analogs that undergo bioreductive activation and thus are targeted to hypoxic tumor cells; 2) Design and synthesis of nucleoside phosphoramidate analogs that can function as prodrugs for nucleotide delivery or as irreversible enzyme inhibitors; 3) Design and synthesis of phosphoramidate analogs targeted to specific DNA sequences; 4) Evaluation of new agents both in vitro and in vivo against established tumor cell lines. Experiments to date have demonstrated that cytotoxic compounds can be prepared in each of these series, and that the compounds are synthetically accessible via techniques currently in use in the laboratory. Antitumor evaluation will rely primarily on the murine B16 system using a clonogenic assay in vitro and using regrowth delay and lung metastases in vivo. Other model systems will be used as needed to assess specific mechanistic objectives. The ultimate goal is to exploit the unique properties of the phosphoramidate alkylating moiety to develop new drugs with reduced host toxicity and/or increases efficacy against resistant or poorly responsive tumors.